1. Field of the Invention
This invention is related to a method for rapidly detecting gene specific methylation through signal amplification using the epi-barcode after hybrid capture of the methylated DNA sequence.
2. Description of the Related Art
DNA methylation is an epigenetic modification which is catalyzed by DNA cytosine-5-methyltransferases (DNMTs) and occurs at the 5-position (C5) of the cytosine ring, within CpG dinucleotides. DNA methylation is essential in regulating gene expression in nearly all biological processes including development, growth, and differentiation (Laird P W et al: Annu. Rev. Genet. 30, 1996; Reik W et al: Science, 293, 2001). Alterations in DNA methylation have been demonstrated to cause a change in the gene expression. For example, hypermethylation leads to gene silencing or the decreased gene expression while hypomethylation activates the genes or increases gene expression. Aberrant DNA methylation is also associated with pathogenesis of diseases such as cancer, autoimmune disorders, and schizophrenia (Baylin S B et al: Nature Clin Pract Oncol Suppl 1, 2005. Richardson B et al: Clin Immunol, 109, 2003. Grayson D R et al: Proc Natl Acad Sci USA, 102, 2005). Most of the current evidences showed that at least in cancer, aberrant DNA methylation could serve a similar function to genetic abnormalities such as inactivating mutations or deletions that lead to abnormal silencing of normal tumor suppressor functions (Garinis G A et al: Hum Genet, 111, 2002). Thus gene/region-specific analysis of DNA methylation could provide valuable information for discovering epigenetic markers used for disease diagnosis, and potential targets used for therapeutics. Furthermore, the methods based on the DNA methylation detection would be highly potential to become disease diagnostic tools.
Many methods for the gene/sequence-specific detection of DNA methylation have been developed over the past decade. Most of these methods are involved in bisulfite conversion of DNA followed by PCR since bisulfite conversion of DNA distinguishes methylated from unmethylated cytosines and enables amplification of DNA methylation to be more specific and reliable. These methods include well-established methylation-specific PCR (MS-PCR), bisulfite sequencing, combined bisulfite restriction analysis (COBRA), and oligonucleotide based microarray (Herman J G et al: Proc Natl Acad Sci USA, 93, 1996; Xiong Z et al: Nucleic Acids Res, 25, 1997; Schumacher A et al: Nucleic Acids Res, 34, 2006). Recently, technically improved methods which are based on the above well-established methods have also been developed. These improved assays enable the detection of DNA methylation to be more specific and quantitative. These assays include real-time methylation-specific PCR such as MethyLight, MethylQuant and QAMA; enzymatic regional methylation assay (ERMA), methylation-specific single-nucleotide primer extension (MS-SNuPE); quantitative bisulfite sequencing using the pyrosequencing technology; and methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) (Eads C A et al: Nucleic Acids Res, 28, 2000; Thomassin H et al: 32, 2004; Zhang Z et al: Anal Chem, 76, 2004; Gonzalgo M L et al: Nucleic Acids Res, 25, 1997; Collella S et al: Biotechnes, 35, 2003; Nygren A O et al: Nucleic Acids Res, 33, 2005). However most of these methods are labor intensive, time-consuming, and/or require large amounts of DNA (>250 ng) as the starting material for DNA modification and amplification. Additionally, as target amplification methods, they need enzymes in the amplification reaction which often results in inhibition of the amplification reaction due to the reduction or loss of enzymes caused by the sample, reaction components or contaminants. They also require the strict reaction conditions which include preparation of highly pure nucleic acid, the complicated design of primer and probe and use of routinely unavailable equipment. Furthermore, they require liquid phase reaction process which in general, can not enable these methods to be configured for solid phase format such as microarray requiring the generation of localized signals at specific locations. The methylation microarray provides an approach for the massively parallel detection of DNA methylation markers. However reliability of this method is severely limited by the complicated sample preparation process before microarray analysis. This off-chip process involves methylation-sensitive enzyme digestion of DNA or immunoprecipitation of methylated DNA, preamplification of the methylated DNA by PCR and sample labeling, which are time consuming and result in poor reproducibility or low accuracy for the assay. Furthermore, the large amount of DNA for an assay is also required by this method.
Because of these shortcomings, the existing technologies for the detection and quantification of the gene-specific methylation are still not satisfying for the routine application in the biomedical field such as disease diagnosis, food and drug industry monitoring, and environmental monitoring. Thus, there is a need for developing a method to improve the detection and quantification of the gene-specific methylation patterns.